A heterobifunctional cross-linking reagent is a molecular substance containing two different types of reactive functional groups that are compatible, i.e., the groups chosen do not cross-react, so that a cross-linking reagent containing them is a relatively stable molecule. Such a reagent can be represented by the designation F.sub.1 -B-F.sub.2 where F.sub.1 represents one or more of a first functional group, F.sub.2 similarly represents one or more of a second functional group that is different from F.sub.1, and B is the backbone of the molecule.
The basic purpose of a F.sub.1 -B-F.sub.2 reagent is to form a covalent linkage between two different molecular components. If the two molecular components are on the same substrate molecule, then an intramolecular cross-link is achieved. If the two molecular components are on different substrate molecules, then the cross-linking is intermolecular, i.e., the two substrate molecules (e.g., S.sub.1 and S.sub.2) are covalently joined together by the cross-linking reagent, forming "S.sub.1 -F.sub.1 -B-F.sub.2 -S.sub.2," which can be abbreviated "S.sub.1 -S.sub.2."
For both intramolecular and intermolecular cross-linking, two types of F.sub.1 -B-F.sub.2 reagents can be considered. The first is a cleavable F.sub.1 -B-F.sub.2, in which the linkage formed by F.sub.1 -B-F.sub.2 on a substrate molecule, or between two substrate molecules, can be reversed under relatively mild, specific conditions. Such conditions, by definition, essentially preserve the structure and/or function of the substrate molecule(s). The second kind of F.sub.1 -B-F.sub.2 reagent forms a noncleavable linkage with respect to subsequent exposure of the linkage to mild, specific conditions.
Both cleavable and noncleavable F.sub.1 -B-F.sub.2 are useful, depending on the application. For example, an "antibody-F.sub.1 -B-F.sub.2 -enzyme" reagent may be formed for use in an immunoassay. If it is important to keep the enzyme attached to the antibody throughout the assay, then a noncleavable F.sub.1 -B-F.sub.2 reagent would be selected. For immunotherapy, an "antibody-F.sub.1 -B-F.sub.2 -toxin" therapeutic agent may be formed in which it is desirable for the toxin to be released at some point from the antibody. In this case, a cleavable F.sub.1 -B-F.sub.2 reagent would be selected.
The general usefulness of an F.sub.1 -B-F.sub.2 reagent is to serve as a molecular tool to combine the properties of substrate molecules. Largely, the applications are biomedical (e.g., diagnostic or therapeutic) or environmental, in which biological molecules like proteins and nucleic acids are at least one of the substrates. Other common substrates, especially for intermolecular cross-linking to one of these two species, are drugs, radionuclides, lipids, carbohydrates, enzyme inhibitors, haptens, signal groups (e.g., fluorophores), metal-chelating groups, and toxins. Also, F.sub.1 -B-F.sub.2 reagents are used to immobilize these kinds of molecules to a solid support such as a chromatographic particle or to the well of a microtiter plate. The resulting, insolubilized substrate then might be used for such applications as affinity chromatography or ligand assay.
F.sub.1 -B-F.sub.2 reagents are part of a larger class of cross-linking reagents that include homobifunctional and zero-length cross-linkers. The homobifunctional reagents possess only a single type of reactive functional group, and the zero-length reagents directly join two existing, functional groups on the substrate molecule(s) without becoming part of the link that develops. The latter are also more conventionally called "activating agents."
The advantage of the F.sub.1 -B-F.sub.2 cross-linkers relative to the homobifunctional and zero-length cross-linkers is the greater control that the F.sub.1 -B-F.sub.2 reagents can provide over the outcome of an intermolecular cross-linking event. Cross-linking with homobifunctional and zero-length cross-linkers tends to be random and produce undesirable side products. For example, the desired coupling of substrate S.sub.1 to S.sub.2 with a homobifunctional or zero-length cross-linker will often lead to the formation of S.sub.1 -S.sub.1 and S.sub.2 -S.sub.2 undesired products in addition to the desired S.sub.1 -S.sub.2 product. Also, S.sub.1, S.sub.2 and S.sub.1 /S.sub.2 polymers can form. Use of F.sub.1 -B-F.sub.2 allows one to form just the S.sub.1 -S.sub.2 product by establishing the specificity of F.sub.1 for S.sub.1 and the specificity of F.sub.2 for S.sub.2.
Carbohydrates have seen some use as homobifunctional cross-linking reagents. For example, drugs have been conjugated to dextran containing aldehyde, hydrazide, or amino groups (Pietersz, G. A., Bioconjugate Chem. 1:89-95 (1990); Wong, S. S., "Chemistry of Protein Conjugation and Cross-Linking," CRC Press, Boca Raton, (1991)).